Cell Cycle Status Affects Coxsackievirus Replication, Persistence, and Reactivation In Vitro

Abstract

Enteroviral persistence has been implicated in the pathogenesis of several chronic human diseases, including dilated cardiomyopathy, insulin-dependent diabetes mellitus, and chronic inflammatory myopathy. However, these viruses are considered highly cytolytic, and it is unclear what mechanisms might permit their long-term survival. Here, we describe the generation of a recombinant coxsackievirus B3 (CVB3) expressing the enhanced green fluorescent protein (eGFP), which we used to mark and track infected cells in vitro. Following exposure of quiescent tissue culture cells to either wild-type CVB3 or eGFP-CVB3, virus production was very limited but increased dramatically after cells were permitted to divide. Studies with cell cycle inhibitors revealed that cells arrested at the G 1 or G 1 /S phase could express high levels of viral polyprotein and produced abundant infectious virus. In contrast, both protein expression and virus yield were markedly reduced in quiescent cells (i. Coxsackieviruses are members of the picornavirus family and Enterovirus genus, which is subdivided into coxsackieviruses A and B, polioviruses, echoviruses, and other unclassified enteroviruses. Acute coxsackievirus infection can cause diseases ranging from mild (rash and myalgia) to severe (pancreatitis, meningitis, and myocarditis). Unsuspected acute viral myocarditis may lead to the collapse and death of young and vigorous individuals, especially during exertion, from catastrophic dysfunction of the electrical pathways in the heart (5, 62). Although the majority of symptomatic patients recover well from acute myocarditis, inflammatory events may continue or recur and can have serious long-term sequelae; some 10 to 20% of patients with symptomatic enteroviral myocarditis (ϳ20,000 to 40,000/year in the United States) will develop chronic disease, progressing over time (usually years) to dilated cardiomyopathy (DCM) (38, 54), where one or both ventricles dilate and decompensate, with resulting cardiac failure. The prevalence of DCM in the general population is much lower (ϳ0.005%), and a large study showed a strong correlation (P Ͻ 0.001) between prior coxsackievirus infection and DCM (51).The enterovirus most commonly associated with myocarditis is coxsackievirus B3 (CVB3), but the mechanisms underlying viral pathogenicity-especially the ongoing myocarditis sometimes seen long after the clearance of infectious virus-remain obscure. Coxsackieviruses are usually considered highly cytolytic, both in tissue culture and in vivo. However, several enteroviruses can establish long-term persistent infections in tissue culture, perhaps by the emergence of viral variants (8,50,58), and some researchers hypothesize that persistent enteroviral infections may underlie several chronic human diseases. Although this idea remains quite controversial for humans (30,35), slot blot hybridization studies have shown positive signal for coxsackievirus RNA in myocardial biopsy specimens from approximately 45% of patients with myocarditis or DCM compared with none ...

Coxsackievirus Targets Proliferating Neuronal Progenitor Cells in the Neonatal CNS

“…Recent studies from several laboratories, including our own, indicate that cellular activation and proliferation may be required for productive coxsackievirus infection in tissue culture cells (Feuer et al, 2002;Luo et al, 2002), and viral binding to target cells has been found to stimulate cellular activation via the ERK1/2 (extracellular signal-regulated kinase) pathway (presumably through receptor signaling), which may in turn provide a better cellular setting for viral replication (Luo et al, 2002). In vivo studies correlate with these findings.…”

“…First, does CVB3 preferentially target proliferating cells in the neonatal CNS? Such a scenario would support our in vitro data, which showed that CVB3 productively infects dividing cells and can remain latent within quiescent cells (Feuer et al, 2002(Feuer et al, , 2004. To answer this question, we investigated the relationship between viral protein expression and cell division; the latter was examined using antibodies against markers of previous cell division [the nucleoside analog bromodeoxyuridine (BrDU)] and active cell proliferation [the nuclear antigen Ki67 (Kiel 67 antigen)].…”

“…Coxsackievirus distribution in the neonatal CNS coincides with regions that contain BrDU ؉ cells Our laboratory has described the construction of a recombinant CVB3 expressing eGFP (Feuer et al, 2002) and its use to characterize the distribution of CVB3 within the CNS of neonatal mice (Feuer et al, 2003). Soon after infection, viral protein expression was observed in the choroid plexus and in neuronal progenitor cells near the lateral ventricles.…”

“…2 B). We have shown that, in tissue culture, CVB3 protein expression is tightly associated with the cell cycle; cells infected with eGFP-CVB and held at G 0 or G 2 /M expressed very little eGFP, but protein expression resumed when the cells were permitted to cycle (Feuer et al, 2002). Therefore, we anticipated that most eGFP ϩ cells in the RMS would be cycling, and thus would express Ki67.…”

Distinct neural stem cell tropism, early immune activation, and choroid plexus pathology following coxsackievirus infection in the neonatal central nervous system

“…[19][20][21] Our laboratory has previously shown that CVB3 preferentially targets NPSCs in culture and in the neonatal host presumably owing to their undifferentiated status and high proliferative index. 20,[22][23][24] In addition, we described the recruitment of unique nestin þ myeloid cells into the CNS following CVB3 infection. 14 These cells were highly susceptible to infection and assisted in the dissemination of infectious virus into the neurogenic regions of the neonatal CNS at early time points.…”